The objective of our current research is to elucidate the molecular mechanism(s) of tissue- and stage-specific, multi-hormone regulated gene expression using the beta-casein gene as a tool. beta-Casein is the major milk protein synthesized by the mammary gland during lactation. The expression of the mouse beta-casein gene is regulated both positively and negatively by the interaction of various peptide and steroid hormones during mammary gland development. Insulin (I), glucocorticoid (H) and prolactin (PRL) are essential for the induction of casein gene transcription and progesterone and EGF inhibit the induction process. PRL exerts its biological effect via a plasma membrane receptor. PRL receptor (PRL-R) exists in at least two forms: a short form (PRL-RS) and a long form (PRL-RL). In the mouse, cDNAs for PRL-RL and three variants of PRL-RS that diverge in the cytoplasmic domains have been isolated and cloned from the mammary gland and the liver, respectively. The two forms differ only in their cytoplasmic domain. Transfection experiments using cDNAs for PRL-R indicated that PRL-RL but not PRL-RS can transduce a PRL signal to stimulate the transcription of casein gene in Chinese hamster ovary cells. To study the mechanism of PRL-R-mediated casein gene expression in the mammary gland, we investigated the regulation of PRL-RL mRNA and its relationship to beta-casein gene expression during development of the mammary gland in vivo and in vitro. We found that mouse mammary epithelium expresses only PRL-RL mRNA and that its level is increased during lactation in vivo and by the synergistic actions of I, H and PRL in vitro. Furthermore, we showed that both progesterone and EGF, negative regulators of casein gene expression, inhibit PRL-RL gene expression both in vivo and in vitro. These results indicate that regulation of RRL-RL mRNA is an important step in hormonal induction of milk protein gene expression in the mammary gland.